Compositions and methods for treating inflammatory bowel disease using a combination therapy of small molecule inhibitors of c-c chemokine receptor type 9 (ccr9) and anti-alpha4beta7 integrin blocking antibodies

ABSTRACT

Provided herein are compositions, methods and kits for treating inflammatory bowel disease (IBD) such as Crohn&#39;s disease and ulcerative colitis in a mammal in need thereof. The method include administering to a subject with IBD a combination therapy containing a therapeutically effective amount of a chemokine receptor 9 (CCR9) inhibitor compound and a therapeutically effective amount of an anti-α4β7 integrin antibody such as vedolizumab. Also provided herein is a kit containing the CCR9 inhibitor compound and anti-α4β7 integrin antibody.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/929,647, filed May 14, 2020, which is adivisional application of U.S. patent application Ser. No. 16/708,167,filed Dec. 9, 2019, which is a continuation application of U.S. patentapplication Ser. No. 14/875,506, filed Oct. 5, 2015, which claimspriority to U.S. Provisional Application No. 62/060,454, filed Oct. 6,2014, the disclosures of which are hereby incorporated by reference intheir entirety for all purposes.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD) is a group of chronic inflammatoryconditions that affects part or all of the gastrointestinal (GI) tractsuch as the mouth, esophagus, stomach, small intestines, largeintestines (colon), rectum, and anus. IBD includes Crohn's disease (CD),ulcerative colitis (UC), and indeterminate colitis. CD and UC can bedistinguished by clinical, endoscopic and pathological features.

CD is a disease of chronic inflammation that can involve any part of theGI tract. Characteristic symptoms of the disease include severeabdominal pain, frequent diarrhea, rectal bleeding, rectal urgency, andswelling of the lower right abdomen.

UC is a chronic intermittent remitting inflammatory disease of thecolon. The disease is characterized by recurring episodes ofinflammation primarily involving superficial mucosal lesions that extendthrough the rectum and upwards through the colon. Acute episodes arecharacterized by chronic diarrhea or constipation, rectal bleeding,cramping and abdominal pain.

IBD is characterized by inflammation and the infiltration of leukocytessuch as lymphocytes, granulocytes, monocytes and macrophages from theblood to the mucosal or epithelial lining of the intestines. Multipleinflammatory cell types including lymphocytes, neutrophils, macrophagesand dendritic cells contribute to IBD. T lymphocytes, for instance,infiltrate the mucosa of the gastrointestinal tract through coordinatedinteractions between adhesion molecules on the surface of the Tlymphocyte and their cognate ligand on the endothelium. For example,α4β7 integrin which expressed on the surface of some T and B lymphocytesdirects the migration of these cells by binding to one of its ligands,mucosal addressin cell adhesion molecule 1 (MAdCAM-1) on endothelialcells of the GI tract. Chemokine receptors and ligands, e.g., thereceptor CCR9 and its ligand CCL25 also play a role in the migration ofinflammatory cells, e.g., effector memory T helper cells into theintestine epithelium in IBD.

Current therapies for treating IBD include surgery or use of anti-tumornecrosis factor (anti-TNFα) antibodies, e.g., infliximab and adalimumab,aminosalicylates, systemic corticosteroids, immunosuppressants, e.g.,thiopurines and methotrexate, and combinations thereof. Unfortunately,some patients with IBD do not respond to or cannot tolerate such drugtreatments.

In view of the above, it is apparent that effective treatment regimensfor IBD that are able to block multiple pathways and/or multiple celltypes associated with lymphocyte infiltration can be useful for treatingthe disease. The present invention provides such therapies along withpharmaceutical compositions and related methods of treatment.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method of treating orreducing the development of inflammatory bowel disease in a mammal, saidmethod comprising administering a suitable amount of a CCR9 chemokinereceptor inhibitor with an anti-α4β7 integrin blocking antibody. In someembodiments, the inflammatory bowel disease is Crohn's disease (CD) orulcerative colitis (UC).

In some embodiments, the CCR9 chemokine receptor inhibitor is a smallmolecule receptor inhibitor having a molecular weight of less than 1500.The CCR9 small molecule receptor inhibitor can have a molecular weightof about 1495, 1450, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600,500, or less.

In other embodiments, the CCR9 chemokine receptor inhibitor is a smallmolecule receptor inhibitor having a molecular weight of less than 750.The CCR9 small molecule receptor inhibitor can have a molecular weightof about 745, 700, 650, 600, 550, 500, 450, 400, 350, 300 or less.

In some embodiments, the CCR9 small molecule inhibitors provided hereinmay be represented by formula (I) or salts thereof:

where R¹, R², R³, R⁴, R⁵, R⁶, L, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ areas defined below.

In some embodiments, the anti-α4β7 integrin blocking antibody isvedolizumab (ENYVIO®), or a biosimilar, biobetter, or bioequivalentthereof. In other embodiments, the anti-α4β7 integrin blocking antibodyis vedolizumab (ENTYVIO®).

In some embodiments, the CCR9 chemokine receptor inhibitor and theanti-α4β7 integrin blocking antibody are administered in a combinationformulation. In other embodiments, the CCR9 chemokine receptor inhibitorand the anti-α4β7 integrin blocking antibody are administeredsequentially. In yet other embodiments, the CCR9 chemokine receptorinhibitor is administered prior to the anti-α4β7 integrin blockingantibody. In another embodiment, the CCR9 chemokine receptor inhibitoris administered after administration of the anti-α4β7 integrin blockingantibody.

In some embodiments, the CCR9 chemokine receptor inhibitor is a compoundhaving the formula:

In another aspect, the present disclosure provides a composition fortreating or reducing the development of inflammatory bowel disease in amammal, said composition comprising a therapeutically effective amountof a CCR9 chemokine receptor inhibitor, a therapeutically effectiveamount of an anti-α4β7 integrin blocking antibody, and apharmaceutically acceptable carrier or excipient.

In some embodiments, the inflammatory bowel disease is Crohn's disease(CD) or ulcerative colitis (UC).

In some embodiments, the CCR9 chemokine receptor inhibitor is a smallmolecule receptor inhibitor having a molecular weight of less than 1500.In other embodiments, the CCR9 chemokine receptor inhibitor is a smallmolecule receptor inhibitor having a molecular weight of less than 750.The CCR9 chemokine receptor inhibitor compound may be represented byformula (I) or salt thereof, as described herein.

In some embodiments, the anti-α4β7 integrin blocking antibody isvedolizumab (ENTYVIO®), or a biosimilar, biobetter, or bioequivalentthereof. In other embodiments, the anti-α4β7 integrin blocking antibodyis vedolizumab (ENTYVIO®).

In yet another aspect, the present disclosure provides a kit fortreating or reducing the development of inflammatory bowel disease in amammal, said kit comprising a therapeutically effective amount of a CCR9chemokine receptor inhibitor, a therapeutically effective amount of ananti-α4β7 integrin blocking antibody, and instructions for effectiveadministration.

In some embodiments, the CCR9 chemokine receptor inhibitor and theanti-α4β7 integrin blocking antibody are formulated for sequentialadministration. In other embodiments, the CCR9 chemokine receptorinhibitor and the anti-α4β7 integrin blocking antibody are formulatedfor concomitant administration.

In some embodiments, the CCR9 chemokine receptor inhibitor is a smallmolecule receptor inhibitor having a molecular weight of less than 1500.In other embodiments, the CCR9 chemokine receptor inhibitor is a smallmolecule receptor inhibitor having a molecular weight of less than 750.The CCR9 chemokine receptor inhibitor compound may be represented byformula (I) or salt thereof, as described herein. In some instances, thesmall molecule receptor inhibitor is vercirnon (Traficet-EN™) or CCX507.

In some embodiments, the anti-α4β7 integrin blocking antibody isvedolizumab (ENYVIO®), or a biosimilar, biobetter, or bioequivalentthereof. In other embodiments, the anti-α4β7 integrin blocking antibodyis vedolizumab (ENTYVIO®).

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows that a small molecule inhibitor of CCR9 (CCX507)limits the binding of retinoic acid differentiated human T cells toMAdCAM-1. FIG. 1A shows human PBMCs were activated with CD3ε and CD28 inthe presence of retinoic acid and IL-12. Expression of gut-tropicfactors, CCR9 and α4β7, are depicted as a two parameter dot plot. Thepercentage of CCR9⁺ α4β7⁺ cells is shown. FIG. 1B shows data of a staticbinding assay of in vitro differentiated human CCR9⁺ α4β7⁺ T cells.Briefly, CCR9⁺ α4β7⁺ cells were mixed with 500 nM of hCCL25 in thepresence or absence of 1 μM CCX507 and added to MAdCAM-1-Fc coatedplates. The number of cells adhering to MAdCAM-1 under each conditionwas quantified using CyQUANT® and depicted as relative fluorescence unit(RFU).

FIGS. 2A, 2B and 2C illustrate in vivo pharmacodynamic efficacy ofCCX507 in a mouse model of an adoptive T cell transfer. FIG. 2A showsthe experimental design used to determine the in vivo pharmacodynamicrange of CCX507 in a short-term T cell trafficking model. The number ofOT-I derived CD8⁺ intra-epithelial lymphocytes (IELs) in the presence of5-30 mg/kg of CCX507 is shown in FIG. 2B and in the presence of ananti-α4β7 integrin blocking antibody is shown in FIG. 2C, as bar graphs.

FIG. 3 shows an association of human CCR9 gene expression in the ileumand colon. Biopsy samples of patients with Crohn's disease wereobtained. Normalized gene expression was derived from terminal ileum andcolon samples. FIG. 3 depicts a heat map representing the expressionlevels of selected genes compared to the expression level of CCR9.

FIGS. 4A, 4B, 4C and 4D show that the combination of CCX507 and ananti-α4β7 integrin blocking antibody conferred greater protectionagainst colitis compared to mono-therapeutic regimens. FIG. 4A shows thedosing regimens for anti-α4β7 integrin blocking antibodies and anti-TNFαantibodies. FIG. 4B provides representative photos depicting the colonof wild-type mice (FVB) and mice receiving vehicle control (1% HPMC),rat IgG2a isotype control, CCX507, anti-α4β7 integrin blocking antibody,and a combination of CCX507 and anti-α4β7 integrin blocking antibody.Quantitative colon vs. weight ratios for CCX507/anti-α4β7 integrinblocking antibody (FIG. 4C) and CCX507/anti-TNFα blocking antibody (FIG.4D) are shown as scatterplots.

FIGS. 5A and 5B shows results from histological analysis of colon tissuefrom mice. FIG. 5A shows representative images of both the proximal anddistal colon from individual mice, representative of the meanhistopathological score for each group. The combination of CCX507 inaddition to anti-α4β7 antibody is shown in the right panel. FIG. 5Bprovides the sum histological score for all mice in the study.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present disclosure is based, in part, on the unexpected discoverythat a combination therapy of a CCR9 inhibitor, e.g., a small moleculeinhibitor of CCR9, and an antibody against α4β7 integrin can actsynergistically in the treatment of inflammatory bowel disease such asCrohn's disease, ulcerative colitis, and indeterminate colitis. Providedherein are methods, compositions and kits for treating IBD in a subject,e.g., human or animal subject, in need thereof. In some embodiments, themethod includes administering therapeutically effective amounts ofCCX507 and vedolizumab to a subject with IBD to elicit a clinicalresponse or maintain clinical remission in the subject.

II. Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings, unlessotherwise indicated.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The term “inflammatory bowel disease” or “IBD” includes gastrointestinaldisorders such as, e.g., Crohn's disease (CD), ulcerative colitis (UC),indeterminate colitis (IC), and IBD that is inconclusive for CD vs. UC(“Inconclusive”). Inflammatory bowel diseases (e.g., CD, UC, IC, andInconclusive) are distinguished from all other disorders, syndromes, andabnormalities of the gastroenterological tract, including irritablebowel syndrome (IBS). Examples of IBD-related diseases includecollagenous colitis and lymphocytic colitis.

The term “ulcerative colitis” or “UC” refers to a chronic intermittentand relapsing inflammatory bowel disease (IBD) of the colon or largebowel characterized by superficial mucosal lesions that extend throughthe rectum and progress upstream. The different types of ulcerativecolitis are classified according to the location and extent ofinflammation. Examples of UC include, but are not limited to, ulcerativeproctitis, proctosigmoiditis, left-sided colitis, and pan-ulcerative(total) colitis.

The term “Crohn's Disease” or “CD” refers to a disease of chronicinflammation that can involve any part of the gastrointestinal tract.Commonly, the distal portion of the small intestine, i.e., the ileum,and the cecum are affected. In other cases, the disease is confined tothe small intestine, colon, or anorectal region. CD occasionallyinvolves the duodenum and stomach, and more rarely the esophagus andmouth. Examples of UC include, but are not limited to, ileocolitis,ileitis, gastroduodenal Crohn's disease, jejunoileitis, and Crohn's(granulomatous) colitis.

The term “subject,” “individual” or “patient” refers to an animal suchas a mammal, including, but not limited to, primates (e.g., humans),cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and thelike.

The term “C—C chemokine receptor type 9,” “CCR9” or “CCR9 chemokinereceptor” refers to a receptor for the chemokine CCL25 which is alsoknown as TECK and SCYA25. The human CCR9 polypeptide sequence is setforth in, e.g., GenBank Accession Nos. NP_001243298, NP_006632,NP_112477, and XP_011531614. The human CCR9 mRNA (coding) sequence isset forth in, e.g., GenBank Accession Nos. NM_001256369, NM_006641,NM_031200, and XM_011533312.

The term “C—C chemokine receptor 9 inhibitor,” “CCR9 inhibitor” or “CCR9chemokine receptor inhibitor” refers to an inhibitor or antagonist of aCCR9 receptor polypeptide, variants thereof, or fragments thereof.

The term “small molecule inhibitor” refers to a small molecule or lowmolecular weight organic compound that inactivates, inhibits, orantagonizes a target molecule, biomolecule, protein or other biologicalproduct.

The term “α4β7 integrin” refers to an heterodimeric integrin moleculethat contains two distinct chains such as an α4 chain and a β7 chain.The human integrin alpha 4 chain polypeptide sequence is set forth in,e.g., GenBank Accession No. NP_000876. The human integrin alpha 4 chainmRNA (coding) sequence is set forth in, e.g., GenBank Accession No.NM_000885. The human integrin beta 7 chain polypeptide sequence is setforth in, e.g., GenBank Accession Nos. NP_000880, XP_005268908, andXP_005268909. The human integrin beta 7 chain mRNA (coding) sequence isset forth in, e.g., GenBank Accession Nos. NM_000889, XM_05268851 andXM_05268852. It is also referred to as LPAM.

The term “anti-α4β7 integrin blocking antibody” or “anti-α4β7 integrinneutralizing antibody” refers to an antibody or a fragment thereof thatspecifically binds to a α4β7 integrin (the α4β7 integrin heterodimer)polypeptide or a fragment thereof. In some cases, an anti-integrin α4β7blocking antibody blocks the interaction of α4β7 integrin with any oneof its ligands.

The term “biosimilar” refers to a biological product that is highlysimilar to an FDA-approved biological product (reference product) andhas no clinically meaningful differences in terms of pharmacokinetics,safety and efficacy from the reference product.

The term “bioequivalent” refers to a biological product that ispharmaceutically equivalent and has a similar bioavailability to anFDA-approved biological product (reference product). For example,according to the FDA the term bioequivalence is defined as “the absenceof a significant difference in the rate and extent to which the activeingredient or active moiety in pharmaceutical equivalents orpharmaceutical alternatives becomes available at the site of drug actionwhen administered at the same molar dose under similar conditions in anappropriately designed study” (United States Food and DrugAdministration, “Guidance for Industry: Bioavailability andBioequicalence Studies for Orally Administered Drug Products—GeneralConsiderations,” 2003, Center for Drug Evaluation and Research).

The term “biobetter” refers a biological product that is in the sameclass as an FDA-approved biological product (reference product) but isnot identical and is improved in terms of safety, efficacy, stability,etc. over the reference product.

The term “therapeutically effective amount” refers to that amount of thetherapeutic agent sufficient to ameliorate the targeted condition orsymptoms. For example, for the given parameter, a therapeuticallyeffective amount will show an increase or decrease of at least 5%, 10%,15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol.

The term “administering” or “administration” and derivatives thereofrefers to the methods that may be used to enable delivery of agents orcompositions to the desired site of biological action. These methodsinclude, but are not limited to parenteral administration (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular,intravascular, intrathecal, intranasal, intravitreal, infusion and localinjection), transmucosal injection, oral administration, administrationas a suppository, and topical administration. One skilled in the artwill know of additional methods for administering a therapeuticallyeffective amount of a compound of the present invention for preventingor relieving one or more symptoms associated with a disease.

The term “treating” or “treatment” refers to the treating or treatmentof a disease or medical condition (such as inflammation) in a patient,such as a mammal (particularly a human or an animal) which includes:ameliorating the disease or medical condition, i.e., eliminating orcausing regression of the disease or medical condition in a patient;suppressing the disease or medical condition, i.e., slowing or arrestingthe development of the disease or medical condition in a patient; oralleviating the symptoms of the disease or medical condition in apatient. The term encompasses the prophylactic treatment of a disease asto prevent or reduce the risk of acquiring or developing a specificdisease, or to prevent or reduce the risk of disease recurrence.

“Alkyl” by itself or as part of another substituent refers to ahydrocarbon group which may be linear, cyclic, or branched or acombination thereof having the number of carbon atoms designated (i.e.,C₁₋₈ means one to eight carbon atoms). The term “cycloalkyl” by itselfor as a part of another substituent refers to a cyclic alkyl grouphaving the number of carbons designated and is a subset of the term“alkyl.” Other subsets of the term “alkyl” include “linear” and“branched” alkyl groups which refer to two different types of acyclicalkyl groups. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,cyclopentyl, (cyclohexyl)methyl, cyclopropylmethyl,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. In this list ofexamples, the methyl, ethyl, n-propyl, and n-butyl alkyl examples arealso examples of “linear alkyl” groups. Similarly, isopropyl and t-butylare also examples of “branched alkyl” groups. Cyclopentyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane are examples of “cycloalkyl” groups. In someembodiments, cyclopropyl may be used as a bridging group between twoother moieties and represeneted as —CH(CH₂)CH—. “Alkyl groups can besubstituted or unsubstituted, unless otherwise indicated. Examples ofsubstituted alkyl include haloalkyl, thioalkyl, aminoalkyl, and thelike. Additional examples of suitable substituted alkyl include, but arenot limited to, hydroxy-isopropyl, —C(CH₃)₂—OH, aminomethyl,2-nitroethyl, 4-cyanobutyl, 2,3-dichloropentyl, and3-hydroxy-5-carboxyhexyl, 2-aminoethyl, pentachloroethyl,trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl,ethoxycarbonylmethyl, methanylsulfanylmethyl, methoxymethyl,3-hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl, and pentafluoroethyl.Suitable substituents for substituted alkyl, include halogen, —CN,—CO₂R′, —C(O)R′, —C(O)NR′R″, oxo (═O or —O⁻), —OR′, —OC(O)R′,—OC(O)NR′R″—NO2, —NR′C(O)R″, —NR′″C(O)NR′R″, —NR′R″, —NR′CO₂R″,—NR'S(O)R″, —NR'S(O)₂R′″, —NR′″S(O)NR′R″, —NR′″S(O)₂NR′R″, —SR′,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′— C(NHR″)═NR′″, —SiR′R″R′″,—OSiR′R″R′″, —N₃, substituted or unsubstituted C₆₋₁₀ aryl, substitutedor unsubstituted 5- to 10-membered heteroaryl, and substituted orunsubstituted 3- to 10-membered heterocyclyl. The number of possiblesubstituents range from zero to (2m′+1), where m′ is the total number ofcarbon atoms in such radical. With respect to substituted alkyl, R′, R″and R′″ each independently refer to a variety of groups includinghydrogen, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₂₋₈ alkenyl, substituted or unsubstituted C₂₋₈ alkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted arylalkyl, substituted or unsubstituted aryloxyalkyl. WhenR′ and R″ are attached to the same nitrogen atom, they can be combinedwith the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring (forexample, —NR′R″ includes 1-pyrrolidinyl and 4-morpholinyl). Furthermore,R′ and R″, R″ and R′″, or R′ and R′″ may together with the atom(s) towhich they are attached, form a substituted or unsubstituted 5-, 6-, or7-membered ring.

“Alkoxy” refers to —O-alkyl. Examples of an alkoxy group includemethoxy, ethoxy, n-propoxy etc.

“Alkenyl” refers to an unsaturated hydrocarbon group which may belinear, cyclic or branched or a combination thereof. Alkenyl groups with2-8 carbon atoms are preferred. The alkenyl group may contain 1, 2 or 3carbon-carbon double bonds. Examples of alkenyl groups include ethenyl,n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl, cyclohexenyl,cyclopentenyl and the like. Alkenyl groups can be substituted orunsubstituted, unless otherwise indicated.

“Alkynyl” refers to an unsaturated hydrocarbon group which may belinear, cyclic or branched or a combination thereof. Alkynyl groups with2-8 carbon atoms are preferred. The alkynyl group may contain 1, 2 or 3carbon-carbon triple bonds. Examples of alkynyl groups include ethynyl,n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. Alkynyl groups canbe substituted or unsubstituted, unless otherwise indicated.

“Alkylamino” refers to —N(alkyl)₂ or —NH(alkyl). When the alkylaminogroup contains two alkyl groups, the alkyl groups may be combinedtogether to form a carbocyclic or heterocylic ring. It is to beunderstood that the alkyl groups of the alkylamino group may besubstituted or unsubstituted. Examples of an alkylamino group includemethylamino, tert-butylamino, dimethylamino, di-isopropylamino,morpholino, and the like.

“Aminoalkyl”, as a substituted alkyl group, refers to a monoaminoalkylor polyaminoalkyl group, most typically substituted with from 1-2 aminogroups. Examples include aminomethyl, 2-aminoethyl, 2-diethylaminoethyl,and the like.

“Aryl” refers to a polyunsaturated, aromatic hydrocarbon group having asingle ring (bicyclic) or multiple rings (preferably bicyclic) which canbe fused together or linked covalently. Aryl groups with 6-10 carbonatoms are preferred, where this number of carbon atoms can be designatedby C₆₋₁₀, for example. Examples of aryl groups include phenyl andnaphthalene-1-yl, naphthalene-2-yl, biphenyl and the like. Aryl groupscan be substituted or unsubstituted, unless otherwise indicated.Substituted aryl may be substituted with one or more substituents.Suitable substituents for aryl include substituted or unsubstituted C₁₋₈alkyl and those substituents as discussed above for substituted alkyl

“Halo” or “halogen”, by itself or as part of a substituent refers to achlorine, bromine, iodine, or fluorine atom.

“Haloalkyl”, as a substituted alkyl group, refers to a monohaloalkyl orpolyhaloalkyl group, most typically substituted with from 1-3 halogenatoms. Examples include 1-chloroethyl, 3-bromopropyl, trifluoromethyland the like.

“Heterocyclyl” refers to a saturated or unsaturated non-aromatic groupcontaining at least one heteroatom (typically 1 to 5 heteroatoms)selected from nitrogen, oxygen or sulfur. Preferably, these groupscontain 0-5 nitrogen atoms, 0-2 sulfur atoms and 0-2 oxygen atoms. Morepreferably, these groups contain 0-3 nitrogen atoms, 0-1 sulfur atomsand 0-1 oxygen atoms. Examples of heterocycle groups includepyrrolidine, piperidine, imidazolidine, pyrazolidine, butyrolactam,valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide,piperidine, 1,4-dioxane, morpholine, thiomorpholine,thiomorpholine-S-oxide, thiomorpholine-S,S-dioxide, piperazine, pyran,pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran,tetrahydrothiophene, quinuclidine and the like. Preferred heterocyclicgroups are monocyclic, though they may be fused or linked covalently toan aryl or heteroaryl ring system.

Exemplary heterocyclic groups may be represented by formula (AA) below:

where formula (AA) is attached via a free valence on either M¹ or M²; M¹represents 0, NR^(e), or S(O)¹; M² represents CR^(f)R^(g), O, S(O)¹, orNR^(e); where it may be necessary to omit one R^(f), R^(g), or R^(e) tocreate a free valence on M¹ or M² such as, for example CR^(f), CR^(g),or N; 1 is 0, 1 or 2; j is 1, 2 or 3 and k is 1, 2 or 3, with theproviso that j+k is 3, 4, or 5; and R^(a), R^(b), R^(c), R^(d), R^(e),R^(f), and R^(g) are independently selected from the group consisting ofhydrogen, halogen, unsubstituted or substituted C₁₋₈ alkyl,unsubstituted or substituted C₂₋₈ alkenyl, unsubstituted or substitutedC₂₋₈ alkynyl, —COR^(h), —CO₂R^(h), —CONR^(h)R^(i), —NR^(h)COR^(i),μSO₂R^(h)—SO₂NR^(h)R^(i), —NR^(h)SO₂R^(i)—NR^(h)R^(i), —OR^(h),—SiR^(h)R^(i)R^(j), —OSiR^(h)R^(i)R^(j), -Q¹COR^(h), -Q¹CO2R^(h),-Q¹CONR^(h)R^(i), -Q¹NR^(h)COR^(i), -Q¹SO₂R^(h), -Q¹SO₂NR^(h)R^(i),-Q¹NR^(h)SO₂R^(i), -Q¹NR^(h)R^(i), -Q¹OR^(h), wherein Q¹ is a memberselected from the group consisting of C₁₋₄ alkylene, C₂₋₄ alkenylene andC₂₋₄ alkynylene, and R^(h), R^(i) and R^(j) are independently selectedfrom the group consisting of hydrogen and C₁₋₈ alkyl, and wherein thealiphatic portions of each of the R^(a), R^(b), R^(c), R^(d), R^(e),R^(f), R^(g), R^(h), R^(i) and R substituents are optionally substitutedwith from one to three members selected from the group consisting ofhalogen, —OH, —OR^(n), —OC(O)NHR^(n), —OC(O)NR^(n)R^(o), μSH, μSR^(n),—S(O)R^(n), —S(O)₂R^(n)—S(O)₂NHR^(n), —S(O)₂NR^(n)R^(o), —NHS(O)₂R^(n),—NR^(n)S(O)₂R^(o), —C(O)NH₂, —C(O)NHR^(n), —C(O)NR^(n)R^(o), —C(O)R^(n),—NHC(O)R^(o), —NR^(n)C(O)R^(o), —NHC(O)NH₂, —NR^(n)C(O)NH₂,—NR^(n)C(O)NHR^(o), —NHC(O)NHR^(n), —NR^(n)C(O)NR^(o)R^(p),—NHC(O)NR^(n)R^(o), —CO₂H, —CO₂R^(n), —NHCO₂R^(n), —NR^(n)CO₂R^(o), —CN,—NO₂, —NH₂, —NHR^(n), —NR^(n)R^(o), —NR^(n)S(O)NH₂ and—NR^(n)S(O)₂NHR^(o), where R^(n), R^(o) and R^(p) are independently anunsubstituted C₁₋₈ alkyl. Additionally, any two of R^(a), R^(b), R^(c),R^(d), R^(e), R^(f), and R^(g) may be combined to form a bridged orspirocyclic ring system.

Preferably, the number of R^(a)+R^(b)+R^(c)+R^(d), groups that are otherthan hydrogen is 0, 1 or 2. More preferably, R^(a), R^(b), R^(c), R^(d),R^(e), R^(f), and R^(g) are independently selected from the groupconsisting of hydrogen, halogen, unsubstituted or substituted C₁₋₈alkyl, —C(O)R^(h), —CO₂R^(h), —C(O)NR^(h)R^(h), —NR^(h)COR^(i),—SO₂R^(h), —SO₂NR^(h)R^(i), —NSO₂R^(h)R^(i), —NR^(h)R^(i), and —OR^(h),where R^(h) and R^(i) are independently selected from the groupconsisting of hydrogen and unsubstituted C₁₋₈ alkyl; and where thealiphatic portions of each of the R^(a), R^(b), R^(c), R^(d), R^(e),R^(f), and R^(g) substituents are optionally substituted with from oneto three members selected from the group consisting of halogen, —OH,—OR^(n), —OC(O)NHR^(n), —OC(O)NR^(n)R^(o), —SH, —SR^(n), —S(O)R^(o),—SO₂R^(n), —SO₂NH₂, —SO₂NHR^(n), —SO₂NR^(n)OR^(o), —NHSO₂R^(n),—NR^(n)SO₂R^(o), —C(O)NH₂, —C(O)NHR^(n), —C(O)NR^(n)R^(o), —C(O)R^(n),—NHC(O)R^(n), —NR^(n)C(O)R^(o), —NHC(O)NH₂, —NR^(n)C(O)NH₂,—NR^(n)C(O)NHR^(o), —NHC(O)NHR^(n), —NR^(n)C(O)NR^(n)R^(o),—NHC(O)NR^(n)R^(o), —CO₂H, —CO₂R^(n), —NHCO₂R^(n), —NR^(n)CO₂R^(o), μCN,μNO₂, —NH₂, —NHR^(n), μNR^(n)R^(o), —NR^(n)S(O)NH₂ and—NR^(n)S(O)₂NHR^(o), where R^(n), R^(o) and R^(p) are independently anunsubstituted C₁₋₈ alkyl.

More preferably, R^(a), R^(b), R^(c), R^(d), R^(e), R^(f), and R^(g) areindependently hydrogen or C₁₋₄ alkyl. In another preferred embodiment,at least three of R^(a), R^(b), R^(c), R^(d), R^(e), R^(f), and R^(g)are hydrogen.

“Heteroaryl” refers to an aromatic group containing at least oneheteroatom. Examples include pyridyl, pyridazinyl, pyrazinyl,pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl,cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl,benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl,isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl,thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines,benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolyl,isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl,triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl,pyrrolyl, thiazolyl, furyl or thienyl. Preferred heteroaryl groups arethose having at least one aryl ring nitrogen atom, such as quinolinyl,quinoxalinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl,benzothiazolyl, indolyl, quinolyl, isoquinolyl and the like. Preferred6-ring heteroaryl systems include pyridyl, pyridazinyl, pyrazinyl,pyrimidinyl, triazinyl and the like. Preferred 5-ring heteroaryl systemsinclude isothiazolyl, pyrazolyl, imidazolyl, thienyl, furyl, triazolyl,tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl,thiazolyl and the like.

Heterocyclyl and heteroaryl can be attached at any available ring carbonor heteroatom. Each heterocyclyl and heteroaryl may have one or morerings. When multiple rings are present, they can be fused together orlinked covalently. Each heterocyclyl and heteroaryl must contain atleast one heteroatom (typically 1 to 5 heteroatoms) selected fromnitrogen, oxygen or sulfur. Preferably, these groups contain 0-5nitrogen atoms, 0-2 sulfur atoms and 0-2 oxygen atoms. More preferably,these groups contain 0-3 nitrogen atoms, 0-1 sulfur atoms and 0-1 oxygenatoms. Heterocyclyl and heteroaryl groups can be substituted orunsubstituted, unless otherwise indicated. For substituted groups, thesubstitution may be on a carbon or heteroatom. For example, when thesubstitution is oxo (═O or O⁻), the resulting group may have either acarbonyl (—C(O)—) or a N-oxide (—N⁺—O⁻) or —S(O)— or —S(O)₂—.

Suitable substituents for substituted alkyl, substituted alkenyl, andsubstituted alkynyl include halogen, —CN, —CO₂R′, —C(O)R′, —C(O)NR′R″,oxo (═O or —O⁻), —OR′, —OC(O)R′, —OC(O)NR′R″—NO₂, —NR′C(O)R″,—NR′″C(O)NR′R″, —NR′R″, —NR′CO₂R″, —NR′S(O)₂R′″, —SR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —SiR′R″R′″, —N₃, substituted or unsubstitutedC₆₋₁₀ aryl, substituted or unsubstituted 5- to 10-membered heteroaryl,and substituted or unsubstituted 3- to 10-membered heterocyclyl, in anumber ranging from zero to (2m′+1), where m′ is the total number ofcarbon atoms in such radical.

Suitable substituents for substituted aryl, substituted heteroaryl andsubstituted heterocyclyl include halogen, —CN, —CO₂R′, —C(O)R′,—C(O)NR′R″, oxo (═O or —O⁻), —OR′, —OC(O)R′, —OC(O)NR′R″, —NO₂,—NR′C(O)R″, —NR′C(O)NR″R′″, —NR′R″, —NR′CO₂R″, —NR′S(O)₂R″, —SR′,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′C(NHR″)═NR′″, —SiR′R″R′″, —N₃,substituted or unsubstituted C1-8 alkyl, substituted or unsubstitutedC₂₋₈ alkenyl, substituted or unsubstituted C₂₋₈ alkynyl, substituted orunsubstituted C₆₋₁₀ aryl, substituted or unsubstituted 5- to 10-memberedheteroaryl, and substituted or unsubstituted 3- to 10-memberedheterocyclyl. The number of possible substituents range from zero to thetotal number of open valences on the aromatic ring system.

As used above, R′, R″ and R′″ each independently refer to a variety ofgroups including hydrogen, substituted or unsubstituted C₁₋₈ alkyl,substituted or unsubstituted C₂₋₈ alkenyl, substituted or unsubstitutedC₂₋₈ alkynyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted arylalkyl, and substituted or unsubstitutedaryloxyalkyl. When R′ and R″ are attached to the same nitrogen atom,they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or7-membered ring (for example, —NR′R″ includes 1-pyrrolidinyl and4-morpholinyl). Furthermore, R′ and R″, R″ and R′″, or R′ and R′″ maytogether with the atom(s) to which they are attached, form a substitutedor unsubstituted 5-, 6-, or 7-membered ring.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, where T and U are independently —NR″″—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A′-(CH₂)_(r)—B′—, where A′ and B′ are independently —CH₂—, —O—, —NR″″—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR″″— or a single bond, and r is an integerfrom 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR″″—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R″″ in —NR″″— and —S(O)₂NR″″— is hydrogen orunsubstituted C₁₋₈ alkyl.

“Heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S)and silicon (Si).

“Pharmaceutically acceptable” carrier, diluent, or excipient is acarrier, diluent, or excipient compatible with the other ingredients ofthe formulation and not deleterious to the recipient thereof.

“Pharmaceutically-acceptable salt” refers to a salt which is acceptablefor administration to a patient, such as a mammal (e.g., salts havingacceptable mammalian safety for a given dosage regime). Such salts canbe derived from pharmaceutically-acceptable inorganic or organic basesand from pharmaceutically-acceptable inorganic or organic acids,depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Salts derived from pharmaceutically-acceptable inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, zinc and the like.Salts derived from pharmaceutically-acceptable organic bases includesalts of primary, secondary, tertiary and quaternary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Salts derivedfrom pharmaceutically-acceptable acids include acetic, ascorbic,benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic,fumaric, gluconic, glucuronic, glutamic, hippuric, hydrobromic,hydrochloric, isethionic, lactic, lactobionic, maleic, malic, mandelic,methanesulfonic, mucic, naphthalenesulfonic, nicotinic, nitric, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand the like.

Also included are salts of amino acids such as arginate and the like,and salts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge, S. M., et al, “Pharmaceutical Salts”, J.Pharmaceutical Science, 1977, 66:1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

“Salt thereof” refers to a compound formed when the hydrogen of an acidis replaced by a cation, such as a metal cation or an organic cation andthe like. Preferably, the salt is a pharmaceutically-acceptable salt,although this is not required for salts of intermediate compounds whichare not intended for administration to a patient.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent drug is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. A wide variety ofprodrug derivatives are known in the art, such as those that rely onhydrolytic cleavage or oxidative activation of the prodrug. An example,without limitation, of a prodrug would be a compound of the presentinvention which is administered as an ester (the “prodrug”), but then ismetabolically hydrolyzed to the carboxylic acid, the active entity.Additional examples include peptidyl derivatives of a compound of theinvention.

Prodrugs of the compounds described herein are those compounds thatreadily undergo chemical changes under physiological conditions toprovide the compounds of the present invention. Additionally, prodrugscan be converted to the compounds of the present invention by chemicalor biochemical methods in an ex vivo environment. For example, prodrugscan be slowly converted to the compounds of the present invention whenplaced in a transdermal patch reservoir with a suitable enzyme orchemical reagent.

Prodrugs may be prepared by modifying functional groups present in thecompounds in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compounds. Prodrugsinclude compounds wherein hydroxyl, amino, sulfhydryl, or carboxylgroups are bonded to any group that, when administered to a mammaliansubject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxylgroup respectively. Examples of prodrugs include, but are not limitedto, acetate, formate and benzoate derivatives of alcohol and aminefunctional groups in the compounds of the invention. Preparation,selection, and use of prodrugs is discussed in T. Higuchi and V. Stella,“Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. SymposiumSeries; “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985; and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, each of which arehereby incorporated by reference in their entirety.

The compounds of the invention may be present in the form ofpharmaceutically acceptable metabolites thereof. The term “metabolite”refers to a pharmaceutically acceptable form of a metabolic derivativeof a compound of the invention (or a salt thereof). In some aspects, themetabolite may be a functional derivative of a compound that is readilyconvertible in vivo into an active compound. In other aspects, themetabolite may be an active compound.

The term “acid isosteres” refers to, unless otherwise stated, a groupwhich can replace a carboxylic acid, having an acidic functionality andsteric and electronic characteristics that provide a level of activity(or other compound characteristic such as solubility) similar to acarboxylic acid. Representative acid isosteres include: hydroxamicacids, sulfonic acids, sulfinic acids, sulfonamides, acyl-sulfonamides,phosphonic acids, phosphinic acids, phosphoric acids, tetrazole, andoxo-oxadiazoles.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, bothsolvated forms and unsolvated forms are intended to be encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms(i.e., as polymorphs). In general, all physical forms are equivalent forthe uses contemplated by the present invention and are intended to bewithin the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers (e.g., separate enantiomers)are all intended to be encompassed within the scope of the presentinvention. The compounds of the present invention may also containunnatural proportions of atomic isotopes at one or more of the atomsthat constitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The compounds of the present invention may include a detectable label. Adetectable label is a group that is detectable at low concentrations,usually less than micromolar, probably less than nanomolar and possiblyless than picomolar, and that can be readily distinguished from othermolecules, due to differences in a molecular property (e.g. molecularweight, mass to charge ratio, radioactivity, redox potential,luminescence, fluorescence, electromagnetic properties, bindingproperties, and the like). Detectable labels may be detected byspectroscopic, photochemical, biochemical, immunochemical, electrical,magnetic, electromagnetic, optical or chemical means and the like.

A wide variety of detectable labels are within the scope of the presentinvention, including hapten labels (e.g., biotin, or labels used inconjunction with detectable antibodies such as horse radish peroxidaseantibodies); mass tag labels (e.g., stable isotope labels);radioisotopic labels (including ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P); metalchelate labels; luminescent labels including fluorescent labels (such asfluorescein, isothiocyanate, Texas red, rhodamine, green fluorescentprotein, and the like), phosphorescent labels, and chemiluminescentlabels, typically having quantum yield greater than 0.1; electroactiveand electron transfer labels; enzyme modulator labels includingcoenzymes, organometallic catalysts horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA; photosensitizerlabels; magnetic bead labels including Dynabeads; colorimetric labelssuch as colloidal gold, silver, selenium, or other metals and metal sollabels (see U.S. Pat. No. 5,120,643, which is herein incorporated byreference in its entirety for all purposes), or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) bead labels; and carbonblack labels. Patents teaching the use of such detectable labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; 4,366,241; 6,312,914; 5,990,479; 6,207,392; 6,423,551;6,251,303; 6,306,610; 6,322,901; 6,319,426; 6,326,144; and 6,444,143,which are herein incorporated by reference in their entirety for allpurposes.

Detectable labels are commercially available or may be prepared as knownto one skilled in the art. Detectable labels may be covalently attachedto the compounds using a reactive functional group, which can be locatedat any appropriate position. Methods for attaching a detectable labelare known to one skilled in the art. When the reactive group is attachedto an alkyl, or substituted alkyl chain tethered to an aryl nucleus, thereactive group may be located at a terminal position of an alkyl chain.

III. Detailed Descriptions of Embodiments

A. Treating Inflammatory Bowel Disease with a Combination Therapy

The present disclosure provides methods, compositions and kits based ona combination therapy that includes a CCR9 inhibitor and an anti-α4β7integrin antibody. This therapy is useful for treating IBD such asCrohn's disease (CD) and ulcerative colitis (UC) in a subject. Thepresent invention is based, in part, on the unexpected discovery thatthe synergistic combination of a CCR9 inhibitor and an anti-α4β7integrin antibody is effective at treating IBD.

1. Crohn's Disease

The compositions, methods and kits of the present invention can be usedto a subject with CD, including all types of CD. The combination therapyof a CCR9 inhibitor and an anti-α4β7 integrin antibody can beadministered at an effective amount to induce a clinical response ormaintain clinical remission in a subject with CD. In some embodiments,the combination therapy mitigates, reduces or minimizes the severity ofone or more symptoms of CD.

Symptoms of CD include diarrhea, fever, fatigue, abdominal pain orcramping, blood in stool, mouth sores, reduced appetite, weight loss,and perianal disease. Additional symptoms or characteristics of CD canbe evaluated by endoscopy, e.g., esophagogastroduodenoscopy,colonoscopy, sigmoidoscopy, endoscopic retrogradecholangiopancreatography, endoscopic ultrasound, and balloon endoscopy,and histology of biopsies form the GI tract. The severity of the diseasecan be categorized as mild to moderate, moderate to severe, andsevere/fulminant disease. Additional descriptions about CD found in, forexample, Lichtenstein et al., Am J Gastroenterol, 2009, 104(2):2465-83.

Severity of CD as well as clinical response to combination therapy canbe determined using a clinical index such as the Crohn's DiseaseActivity Index or CDAI (Best et al., Gastroenterology, 1976, 70:439-44).The index is used to quantify the symptoms of patients with CD. The CDAIcan be used to define clinical response or remission of CD. The CDAIconsists of eight factors, each added together (summed) after adjustmentwith a weighting factor or multiplier. The eight factors include numberof liquid stools, abdominal pain, general well-being, extraintestinalcomplications, antidiarrheal drugs, abdominal mass, hemacrit, and bodyweight. Remission of Crohn's disease is generally defined as a fall ordecrease in the CDAI of less than 150 points. Severe disease istypically defined as a value of greater than 450 points. In certainaspects, response to a particular medication in a Crohn's diseasepatient is defined as a fall of the CDAI of greater than 70 points frombaseline (week 0 of treatment).

Clinical index such as the CDAI can be used to determine whether thecombination therapy described herein induces a clinical response orclinical remission in a patient with Crohn's disease. In someembodiments, if the patient's CDAI score decreases by 70 point or morefrom baseline upon receiving the combination therapy, the patient ishaving a clinical response. If the patient's CDAI score decreases toless than 150 points at the end of the induction phase of therapy, thepatient is in clinical remission of CD.

2. Ulcerative Colitis

The compositions, methods and kits of the present invention can be usedto a subject with UC, including all types of UC. The combination therapyof a CCR9 inhibitor and an anti-α4β7 integrin antibody can beadministered at an effective amount to induce a clinical response ormaintain clinical remission in a subject with UC. In some embodiments,the combination therapy mitigates, reduces or minimizes the severity ofone or more symptoms of UC.

Symptoms of UC include, but are not limited to, diarrhea, abdominal painand cramping, rectal pain, rectal bleeding, urgency to have a bowelmovement, inability to have a bowel movement, weight loss, fatigue,fever, or anemia. The severity of the disease can be categorized as mildto moderate, moderate to severe, and severe/fulminant disease. See,e.g., Kornbluth et al., Am J Gastroenterol, 2004, 99(7):1371-85.

Disease activity of UC and response to treatment can be assessed byquantitative analysis using a composite index scoring system. Generally,clinicians consider at least four factors or variables when assessing UCdisease activity: clinical symptoms, quality of life, endoscopyevaluation, and histology assessment. For example, the colitis activityindex (CAI) is a quantitative measurement of incorporates the followingdisease symptoms: inflammation in the colon based on colonoscopy,diarrhea, abdominal pain and cramping, and blood stool. Standardizedendoscopic score systems such as the UC Endoscopic Index of Severity(UCEIS) are useful for establishing a patient's disease index score.Other useful disease activity indices include the Mayo Clinic Score(see, e.g., Rutgeert et al., N Eng J Med, 2005, 353(23):2462-76) and themodified Mayo Disease Activity Index (MMDAI; see, e.g., Schroeder etal., N Eng J Med, 1987, 317(26):1625-9). The four factors used in theMayo Clinic scoring system include stool (bowel) frequency, rectalbleeding, endoscopic findings, and the physician's global assessment ofdisease severity (e.g., daily abdominal discomfort and general sense ofwell-being).

Compared to the Mayo Clinic Score, MMDAI includes the removal of“friability” from the endoscopy score of 1. Therefore, the presence offriability reflects an endoscopy score of 2 or 3. The MMDAI evaluates 4subscores (bowel frequency, rectal bleeding, endoscopic appearance, andphysician's global assessment), each on a scale of 0 to 3 with a maximumtotal score of 12.

In some embodiments, clinical response by a subject with UC to acombination therapy provided herein corresponds to a decrease of 2points or greater from baseline in the MMDAI score and a 25% or greaterdecrease from baseline, and/or a decrease of a 1 point or greater frombaseline in the rectal bleeding subscore. In other embodiments, clinicalresponse corresponds to a decrease of 3 points or greater in Mayo ClinicScore and 30% from baseline

Clinical remission by a UC subject administered the combination therapycan correspond to a score of 0 for rectal bleeding and a combined scoreof 2 point or lower for bowel frequency and physician's assessment usingthe MMDAI subscale. In other embodiments, clinical remission in asubject with UC refers to having a Mayo Clinic Score of 2 point or lessand no individual subscore (bowel frequency, rectal bleeding, endoscopicappearance, and physician's global assessment) of more than 1 point.

B. Combination Therapy of CCR9 Inhibitors and Anti-α4β7 IntegrinAntibodies

Provided herein are methods, compositions and kits that take advantageof the synergistic effect of CCR9 inhibitors and anti-α4β7 integrinantibodies in reducing inflammation in subjects with IBD. A combinationtreatment that includes both a CCR9 inhibitor and an anti-α4β7 integrinantibody is more effective at treating one or more symptoms of IBDcompared to either compound/antibody alone.

1. Chemokine Receptor Type (CCR9) Inhibitors

The present invention provides compounds that modulate CCR9 activity.Specifically, the invention provides compounds having anti-inflammatoryor immunoregulatory activity. The compounds of the invention are thoughtto interfere with inappropriate T-cell trafficking by specificallymodulating or inhibiting a chemokine receptor function. Chemokinereceptors are integral membrane proteins which interact with anextracellular ligand, such as a chemokine, and mediate a cellularresponse to the ligand, e.g., chemotaxis, increased intracellularcalcium ion concentration, etc. Therefore, modulation of a chemokinereceptor function, e.g., interference with a chemokine receptor-ligandinteraction, can inhibit or reduce a chemokine receptor mediatedresponse, as wells as treat or prevent a chemokine receptor mediatedcondition or disease.

Without being bound by any particular theory, it is believed that thecompounds provided herein interfere with the interaction between CCR9and its ligand CCL25. For example, compounds of this invention act aspotent CCR9 antagonists, and this antagonistic activity has been furtherconfirmed in animal testing for inflammation, one of the hallmarkdisease states for CCR9. Compounds contemplated by the inventioninclude, but are not limited to, the exemplary compounds provided hereinand salts thereof.

For example, useful compounds act as potent CCR9 antagonists, and thisantagonistic activity has been further confirmed in animal testing forinflammation, one of the hallmark disease states for CCR9. Accordingly,the compounds provided herein are useful in pharmaceutical compositionsand methods for the treatment of inflammatory bowel disease, e.g.,ulcerative colitis and Crohn's disease.

In some embodiments, CCR9 inhibitors, e.g., CCR9 small moleculeinhibitors of the present disclosure are represented by formula (I), orsalts thereof:

where R¹ is selected from the group consisting of substituted orunsubstituted C₂₋₈ alkyl, substituted or unsubstituted C₁₋₈ alkoxy,substituted or unsubstituted C₁₋₈ alkylamino, and substituted orunsubstituted C₃₋₁₀ heterocyclyl, and;

R² is H, F, Cl, or substituted or unsubstituted C₁₋₈ alkoxy; or

R¹ and R² together with the carbon atoms to which they are attached forma non-aromatic carbocyclic ring or a heterocyclic ring;

R³ is H, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, or halo;

R⁴ is H or F;

R⁵ is H, F, Cl, or CH₃;

R⁶ is H, halo, —CN, —CO₂R^(a), —CONH₂, —NH₂, substituted orunsubstituted C₁₋₈ alkyl, substituted or unsubstituted C₁₋₈ alkoxy, orsubstituted or unsubstituted C₁₋₈ aminoalkyl;

R^(a) is H or substituted or unsubstituted C₁₋₈ alkyl;

where R⁵ and R⁶ may together form a carbocyclic ring;

L is a bond, —CH₂—, or —CH(CH₃)—;

each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selectedfrom the group consisting of N, N—O, and —CR⁸—; where at least one andnot more than two of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are N or N—O;

R⁸ is each independently selected from the group consisting of H, halo,—CN, —OH, oxo, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, and —NR²⁰R²¹, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl; and

R²⁰ and R²¹ are each independently H, or substituted or unsubstitutedC₁₋₈ alkyl.

In some embodiments of formula (I), one of A¹ or A² is N or N—O, and theremaining of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are —CR⁸—, where each R⁸is selected independently.

In some embodiments, two of, A², A³, A⁴, A⁵ is N or N—O, and theremaining of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are —CR⁸—, where each R⁸is selected independently.

In some embodiments, the compounds or compositions of formula (I) arerepresented by formula (II) or salts thereof:

where R¹ is selected from the group consisting of substituted orunsubstituted C₂₋₈ alkyl, substituted or unsubstituted C₁₋₈alkoxy,substituted or unsubstituted C₁₋₈ alkylamino, and substituted orunsubstituted C₃₋₁₀ heterocyclyl;

R² is H, F, Cl, or substituted or unsubstituted C₁₋₈ alkoxy; or

R¹ and R² together with the carbon atoms to which they are attached forma non-aromatic carbocyclic ring or a heterocyclic ring;

R³ is H, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, or halo;

R⁴ is H or F;

R⁵ is H, F, Cl, or —CH₃;

R⁶ is H, halo, —CN, —CO₂R^(a), —CONH₂, —NH₂, substituted orunsubstituted C₁₋₈ aminoalkyl, substituted or unsubstituted C₁₋₈ alkyl,or substituted or unsubstituted C₁₋₈ alkoxy;

R^(a) is H or substituted or unsubstituted C₁₋₈ alkyl;

where R⁵ and R⁶ may together form a carbocyclic ring;

L is a bond, —CH₂—, or —CH(CH₃)—; and

Z is selected from the group consisting of:

and N-oxides thereof;

where the Z group may be unsubstituted or substituted with 1 to 3independently selected R⁸ substituents;

each R⁸ is independently selected from the group consisting of H, halo,—CN, —OH, oxo, substituted or unsubstituted C₁₋₈alkyl, substituted orunsubstituted C₁₋₈ alkoxy, and —NR²⁰R²¹, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl; and

R²⁰ and R²¹ are each independently H, substituted or unsubstituted C₁₋₈alkyl.

In some embodiments of formula (II), Z is selected from the groupconsisting of: substituted or unsubstituted quinolinyl, substituted orunsubstituted isoquinolinyl, substituted or unsubstituted1,6-naphthyridinyl, substituted or unsubstituted cinnolinyl, substitutedor unsubstituted phthalazinyl, substituted or unsubstitutedquinazolinyl.

In one embodiment, the compounds or compositions of formula (I) providedherein are represented by formula (IIIa) or (IIIb), or salts thereof:

where R¹ is selected from the group consisting of substituted orunsubstituted C₂₋₈ alkyl, substituted or unsubstituted C₁₋₈ alkoxy,substituted or unsubstituted C₁₋₈ alkylamino, and substituted orunsubstituted C₃₋₁₀ heterocyclyl;

R² is H, F, Cl, or substituted or unsubstituted C₁₋₈ alkoxy; or

R¹ and R² together with the carbon atoms to which they are attached forma non-aromatic carbocyclic ring or a heterocyclic ring;

R³ is H, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, or halo;

R⁴ is H or F;

R^(s) is H, F, Cl, or CH₃;

R⁶ is H, halo, —CN, —CO₂R^(a), —CONH₂, —NH₂, substituted orunsubstituted C₁₋₈ aminoalkyl, substituted or unsubstituted C₁₋₈ alkyl,or substituted or unsubstituted C₁₋₈ alkoxy;

R^(a) is H or substituted or unsubstituted C₁₋₈ alkyl;

or where R⁵ and R⁶ together with the carbon atoms to which they areattached form a carbocyclic ring;

each R⁸ is independently selected from the group consisting of H, halo,—CN, —OH, oxo, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, and —NR²⁰R²¹, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl;

R²⁰ and R²¹ are each independently H, or substituted or unsubstitutedC₁₋₈ alkyl; and

n is 0, 1, 2 or 3.

In one embodiment of formula (IIIa) or (IIIb) or salts thereof, R¹ isselected from the group consisting of: —CH₂CH₃, —CH(CH₃)₂, —C(CH₃)₃,—C(CH₃)₂CH₂CH₃, —C(CH₂CH₂)CN, —C(OH)(CH₃)₂, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂,—OC(CH₃)₃, —OCH₂CH(CH₃)₂, —OCF₃, and morpholino; R² is H, F, or Cl; orR¹ and R² may together form —OC(CH₃)₂CH₂ or —C(CH₃)₂CH₂CH₂—; R³ is H,—CH₃, or —OCH₃; R⁴ is H or F; R⁵ is H; R⁶ is H, —CH₃, —CH₂CH₃,—CH(CH₃)₂, —C₃H₇, —CH₂F, —CHF₂, —CF₂CH₃, —CF₃, —CH₂OCH₃, —CH₂OH, —CH₂CN,—CN, or —CONH₂; and each R⁸ is independently selected from the groupconsisting of H, F, Cl, Br, —CH₃, —OH, —OCH₃, —OCH₂CH₃, —NH₂, —N(CH₃)₂,and —CN. In some instances, R¹ is —C(CH₃)₃.

In other embodiments of formula (IIIa) or formula (IIIb), R² is H or F;R³ is H; R⁴ is H; and R⁶ is —CH₃, —CH₂F, —CHF₂, or —CF₃.

In one embodiment, the compounds and compositions of formula (IIIa) or(IIIb) or salts thereof are selected from the group consisting of:

In some embodiments, CCR9 inhibitors, e.g., CCR9 small moleculeinhibitor compounds and compositions provided herein are selected fromthe group consisting of:

N-oxides thereof.

In some embodiments, the preferred R¹ substituents are as follows. Informula (I, II, IIIa, and IIIb), R¹ is selected from the groupconsisting of substituted or unsubstituted C₂₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, substituted or unsubstituted C₁₋₈ alkylamino,and substituted or unsubstituted C₃₋₁₀ heterocyclyl. When R¹ issubstituted alkyl, the alkyl group is preferably substituted with haloor hydroxy. When R¹ is substituted alkoxy, the alkoxy group ispreferably substituted with halo. Preferably R¹ is unsubstituted C₂₋₈alkyl, including C₃₋₈ cycloalkyl, C₂₋₈ haloalkyl, C₁₋₈ hydroxyalkyl,unsubstituted C₁₋₈ alkoxy, C₁₋₈ haloalkoxy, and C₁₋₈ alkylamino; morepreferably unsubstituted C₂₋₈ alkyl, C₂₋₈ haloalkyl, unsubstituted C₁₋₈alkoxy, and C₁₋₈ alkylamino; even more preferably unsubstituted C₂₋₈alkyl, unsubstituted C₁₋₈ alkoxy, and morpholino; still more preferablyunsubstituted C₂₋₈; and most preferably t-butyl.

In some embodiments, the preferred R⁶ substituents are as follows. Informula (I, II, IIIa, and IIIb), R⁶ is H, halo, —CN, —CO₂R_(a), —CONH₂,—NH₂, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, or substituted or unsubstituted C₁₋₈aminoalkyl. When R⁶ is substituted alkyl, the alkyl group is preferablysubstituted with halo, hydroxy, alkoxy, or cyano. Preferably R⁶ is —CN,—CONH₂, —NH₂, unsubstituted C₁₋₈ alkyl, unsubstituted C₁₋₈ haloalkyl,and unsubstituted C₁₋₈ alkoxy; more preferably unsubstituted C₁₋₈ alkyl,or unsubstituted C₁₋₈ haloalkyl, even more preferably unsubstituted C₁₋₈alkyl; most preferably methyl.

In some embodiments, the CCR9 small molecule inhibitor compound of thepresent invention is

In one embodiment, the CCR9 small molecule inhibitor is vercirnon(Traficet-EN™) or CCX507.

Detailed descriptions of the CCR9 inhibitor compounds provided hereinand methods for preparing such compounds is found in, for example, U.S.Pat. No. 8,916,601 and U.S. Patent Application Publication Nos.2013/0267492, 2013/0059893, 2012/0245138, 2012/0165303, 2011/0021523,2010/0331302, 2010/0227902, 2010/0190762, 2010/0152186, 2010/0056509,2009/0163498 and 2009/0005410, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

The compounds provided herein may be may be synthesized using a varietyof standard organic chemistry transformations. Certain general reactiontypes employed widely to synthesize target compounds in this inventionare summarized in the examples. Specifically, generic procedures forsulfonamide formation and aza-aryl N-oxide formation are describedwithin and were employed routinely.

While not intended to be exhaustive, representative synthetic organictransformations which can be used to prepare compounds of the inventionare included herein. These representative transformations include;standard functional group manipulations; reductions such as nitro toamino; oxidations of functional groups including alcohols and aza-aryls;aryl substitutions via IPSO or other mechanisms for the introduction ofa variety of groups including nitrile, methyl and halogen; protectinggroup introductions and removals; Grignard formation and reaction withan electrophile; metal-mediated cross couplings including but notlimited to Buckwald, Suzuki and Sonigashira reactions; halogenations andother electrophilic aromatic substitution reactions; diazonium saltformations and reactions of these species; etherifications; cyclativecondensations, dehydrations, oxidations and reductions leading toheteroaryl groups; aryl metallations and transmetallations and reactionof the ensuing aryl-metal species with an electrophile such as an acidchloride or Weinreb amide; amidations; esterifications; nucleophilicsubstitution reactions; alkylations; acylations; sulfonamide formation;chlorosulfonylations; ester and related hydrolyses, and the like.

Certain molecules claimed in this patent can exist in differentenantiomeric and diastereomeric forms and all such variants of thesecompounds are within the scope of the invention. In particular, when R⁸is OH and ortho to a nitrogen, although illustrated by formula as—N═C(OH)— it is to be understood that the tautomeric form —NH—C(O)— isalso within the scope of the formula.

In the descriptions of the syntheses that follow, some precursors wereobtained from commercial sources. These commercial sources includeAldrich Chemical Co., Acros Organics, Ryan Scientific Incorporated,Oakwood Products Incorporated, Lancaster Chemicals, Sigma Chemical Co.,Lancaster Chemical Co., TCI-America, Alfa Aesar, Davos Chemicals, andGFS Chemicals.

2. Pharmaceutical Formulations of CCR9 Inhibitors

In another aspect, the present disclosure provides compositions orformulations that modulate CCR9 activity. Generally, the compositions orformulations for modulating chemokine receptor activity in a subjectsuch as a human or animal will comprise a compound provided herein and apharmaceutically acceptable excipient or diluent.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The pharmaceutical compositions for the administration of the compoundsof this invention may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the pharmaceutical compositions are prepared byuniformly and intimately bringing the active ingredient into associationwith a liquid carrier or a finely divided solid carrier or both, andthen, if necessary, shaping the product into the desired formulation. Inthe pharmaceutical composition the active object compound is included inan amount sufficient to produce the desired effect upon the process orcondition of diseases.

In some embodiments, the CCR9 inhibitor of the present disclosure is apharmaceutical compound having a crystalline form. A non-limitingexample of such a crystalline form of a CCR9 inhibitor is described in,e.g., U.S. Pat. No. 9,133,124, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions and self-emulsifications as described in U.S. Pat. No.6,451,399, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions. Suchcompositions may contain one or more agents selected from sweeteningagents, flavoring agents, coloring agents and preserving agents in orderto provide pharmaceutically elegant and palatable preparations. Tabletscontain the active ingredient in admixture with other non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, inertdiluents such as cellulose, silicon dioxide, aluminum oxide, calciumcarbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose,calcium phosphate or sodium phosphate; granulating and disintegratingagents, for example, corn starch, or alginic acid; binding agents, forexample PVP, cellulose, PEG, starch, gelatin or acacia, and lubricatingagents, for example magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated enterically or otherwiseby known techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in the U.S. Pat. Nos. 4,256,108;4,166,452; and 4,265,874 to form osmotic therapeutic tablets for controlrelease.

Formulations for oral use may also be presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules where the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin, or olive oil.Additionally, emulsions can be prepared with a non-water miscibleingredient such as oils and stabilized with surfactants such asmono-diglycerides, PEG esters and the like.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil in water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative. and flavoring and coloringagents. Oral solutions can be prepared in combination with, for example,cyclodextrin, PEG and surfactants.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, axed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds disclosed herein may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.Additionally, the compounds can be administered viaocular delivery bymeans of solutions or ointments. Still further, transdermal delivery ofthe subject compounds can be accomplished by means of iontophoreticpatches and the like.

For topical use, creams, ointments, jellies, solutions or suspensionscontaining the compounds of the present invention are employed. As usedherein, topical application is also meant to include the use of mouthwashes and gargles.

The pharmaceutical compositions and methods of the present invention mayfurther comprise other therapeutically active compounds as noted herein,such as those applied in the treatment of the above mentionedpathological conditions.

3. Anti-α4β7 Integrin Blocking Antibodies

Anti-α4β7 integrin antibodies suitable for use in the treatment ofinflammatory bowel disease, e.g., Crohn's disease and ulcerative colitisinclude antibodies from any desired source that inhibits the binding ofα4β7 integrin to any one of its ligands such as mucosal addressin(MadCAM-1), fibronectin, vascular cell adhesion molecule (VCAM), and thelike. Anti-α4β7 integrin antibodies can be human antibodies, mouseantibodies, rabbit antibodies, engineered antibodies such as chimericantibodies, humanized antibodies, and antigen-binding fragments ofantibodies such as Fab, Fv, scFv, Fab′ and F(ab′)₂ fragments.

In some embodiments, the anti-α4β7 integrin antibody binds to an epitopeon the α4 chain, an epitope on the β7 chain, or a composite epitopeformed by the association of the α4 chain and the β7 chain. In somecases, the anti-α4β7 integrin antibody binds an epitope formed by theassociation of the α4 and β7 chains, and does not bind an epitope on theα4 chain or on the β7 chain, if the α4 and β7 chain are not associatedor complexed together.

Non-limiting examples of an anti-α4β7 integrin antibody for use in themethod described herein include vedolizumab (ENTYVIO®), natalizumab(Tysabri©), AMG 181 (Amgen) and those described in, e.g., InternationalPatent Publication No. WO 2012/151248, U.S. Pat. Nos. 7,147,851;7,402,410; 8,444,981; 8,454,961; 8,454,962; 8,871,490; and U.S. PatentApplication Publication No. 2015/0086563, the disclosures of which areherein incorporated by reference in their entirety for all purposes.Additional useful anti-α4β7 integrin antibodies include bioequivalents,biosimilars, and biobetters of any of the anti-α4β7 integrin antibodiesdescribed herein.

In some embodiments, the anti-α4β7 integrin antibody of the presentdisclosure is an antibody with an amino acid sequence that has at least70%, at least 80%, at least 90%, at least 95% or more sequence identityto an anti-α4β7 integrin reference antibody such as vedolizumab, AMG 181or other anti-α4β7 integrin antibody that is known to one skilled in theart. In some instances, the antibody variant has one or more amino acidsubstitutions, deletions and/or additions at certain amino acidpositions of the reference antibody, but retains antigen bindingactivity.

One of skill in the art recognizes that “percent of sequence identity”can determined by comparing two optimally aligned sequences over acomparison window or designated region of the sequence, wherein theportion of the polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage can be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Percent sequence identity can be measured using a BLAST orBLAST 2.0 sequence comparison algorithms, with default parameters, or bymanual alignment and visual inspection.

Antibodies, fragments thereof, variants thereof and derivatives thereofmay be generated using a variety of standard methods recognized by thoseskilled in the art. See, e.g., Harlow, E. and Lane D P. Antibodies: ALaboratory Manual. Cold Spring Harbor: Cold Spring Harbor LaboratoryPress, 1988. Antigen-binding fragments such as Fab and F(ab′)₂ fragmentsmay be produced by genetic engineering. Procedures for the production ofchimeric and further engineered monoclonal antibodies include thosedescribed in Riechmann et al., Nature, 1988, 332:323, Liu et al., Proc.Nat. Acad. Sci. USA, 1987, 84:3439, Larrick et al., Bio/Technology,1989, 7:934, and Winter et al., TIPS, 1993, 14:139. Examples oftechniques for production and use of transgenic animals for theproduction of human or partially human antibodies are described in,e.g., Davis et al., 2003, Production of human antibodies from transgenicmice in Lo, ed. Antibody Engineering Methods and Protocols, HumanaPress, NJ: 191-200.

4. Pharmaceutical Formulations of Anti-α4β7 Integrin Antibodies

Provided herein are formulations of the anti-α4β7 integrin antibody thatcan stabilize the antibody, reduce the formation of antibody aggregates,retard the degradation of the antibody, and/or minimize theimmunogenicity of the antibody. The formulation can include anantioxidant or chelator, at least one free amino acid, a surfactant, anon-reducing sugar, and/or a buffering agent.

The antioxidant or chelator can be citrate, ethylenediaminetetraaceticacid (EDTA), ethyleneglycoltetraacetic acid (EGTA), dimercaprol,diethylenetriaminepentaacetic acid, or N,N-bis(carboxymethyl)glycine;preferably citrate or EDTA. The free amino acid can be histidine,alanine, arginine, glycine, glutamic acid and combinations thereof. Thesurfactant can be polysorbates 20; polysorbate 80; TRITON(t-octylphenoxypolyethoxyethanol, nonionic detergent; sodium dodecylsulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; sorbitan monopalmitate; polyethyl glycol (PEG),polypropylene glycol (PPG), and copolymers of poloxyethylene andpoloxypropylene glycol; preferably polysorbates 80.

The buffering agent can be a buffer that can adjust the pH of theformulation to about 5.0 to about 7.5, to about pH 5.5 to about 7.5, toabout pH 6.0 to about 7.0, or to a pH of about 6.3 to about 6.5.Non-limiting examples of a buffering agent include acetate, succinate,gluconate, histidine, citrate, phosphate, maleate, cacodylate,2-[N-morpholino]ethanesulfonic acid (MES),bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris),N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine and otherorganic acid buffers, preferably histidine or citrate.

In some embodiments, the anti-α4β7 integrin antibody is in a lyophilizedformulation, e.g., a dry form. In some cases, the lyophilizedformulation includes the anti-α4β7 integrin antibody and one or moreexcipients, such as a non-reducing sugar, a buffering agent, a freeamino acid, and/or a surfactant.

In some cases, the lyophilized formulation contains at least about 50mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, atleast about 90 mg, at least about 100 mg, at least about 120 mg, atleast about 140 mg, at least about 180 mg, at least about 200 mg, atleast about 220 mg, at least about 240 mg, at least about 280 mg, atleast about 300 mg, at least about 400 mg, at least about 500 mg, atleast about 600 mg, at least about 700 mg, at least about 800 mg, atleast about 900 mg of anti-α4β7 integrin antibody. In some cases, thelyophilized formulation is stored as a single dose in one vial.

In some embodiments, the anti-α4β7 integrin antibody is a liquidformulation. Such a formulation can include the anti-α4β7 integrinantibody, a buffering agent, a non-reducing sugar, and/or a free aminoacid.

The amount of antibody present in a liquid formulation can be at leastabout 25 mg/ml to about 200 mg/ml anti-α4β7 integrin antibody, e.g., 25mg/ml to about 200 mg/ml, 25 mg/ml to about 150 mg/ml, 25 mg/ml to about100 mg/ml, 50 mg/ml to about 200 mg/ml, 50 mg/ml to about 150 mg/ml, 50mg/ml to about 100 mg/ml, 100 mg/ml to about 200 mg/ml, or 150 mg/ml toabout 200 mg/ml anti-α4β7 integrin antibody.

The non-reducing sugar can be, but not limited to, mannitol, sorbital,sucrose, trehalose, raffinose, stachyose, melezitose, dextran, maltitol,lactitol, isomaltulose, palatinit and combinations thereof. In someembodiments, the ratio of the non-reducing sugar to the anti-α4β7integrin antibody is at least 400:1 (mole:mole), at least 400:1(mole:mole), at least 400:1 (mole:mole), at least 600:1 (mole:mole), atleast 625:1 (mole:mole), at least 650:1 (mole:mole), at least 700:1(mole:mole), at least 750:1 (mole:mole), at least 800:1 (mole:mole), atleast 1000:1 (mole:mole), at least 1100:1 (mole:mole), at least 1200:1(mole:mole), at least 1300:1 (mole:mole), at least 1400:1 (mole:mole),at least 1500:1 (mole:mole), at least 1600:1 (mole:mole), at least1700:1 (mole:mole), at least 1800:1 (mole:mole), at least 1900:1(mole:mole), or at least 2000:1 (mole:mole).

Exemplary formulations of the anti-α4β7 integrin antibody include, butare not limited, those described in, e.g., U.S. Patent ApplicationPublication Nos. 2012/0282249, 2014/0377251; and 2014/0341885, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

C. Methods of Administration of Combination Therapy

In another aspect, the present disclosure provides a combination therapyfor the treatment of IBD, e.g., CD and UC. The combination therapyincludes a therapeutically effective amount of a CCR9 inhibitor and atherapeutically effective amount of an anti-α4p 7 integrin blockingantibody. The combination of therapeutic agents can act synergisticallyto effect the treatment or prevention of the various disorders. Usingthis approach, therapeutic efficacy can be achieved using lower dosagesof each agent, thus reducing the potential for adverse side effects.

The term “therapeutically effective amount” means the amount of thesubject compound that will elicit the biological or medical response ofa cell, tissue, system, or animal, such as a human, that is being soughtby the researcher, veterinarian, medical doctor or other treatmentprovider.

Depending on the disease status and the subject's condition, thecompounds, antibodies, and formulations of the present disclosure may beadministered by oral, parenteral (e.g., intramuscular, intraperitoneal,intravenous, ICV, intracisternal injection or infusion, subcutaneousinjection, or implant), inhalation, nasal, vaginal, rectal, sublingual,or topical routes of administration. In addition, the compounds andantibodies may be formulated, alone or together, in suitable dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants and vehicles appropriate for each rouseof administration. The present disclosure also contemplatesadministration of the compounds and antibodies of the present disclosurein a depot formulation.

In the treatment of IBD such as Crohn's disease and UC, an appropriatedosage level of a CCR9 inhibitor will generally be about 0.001 to 100 mgper kg patient body weight per day which can be administered in singleor multiple doses. Preferably, the dosage level will be about 0.01 toabout 50 mg/kg per day; more preferably about 0.05 to about 10 mg/kg perday. A suitable dosage level may be about 0.01 to 50 mg/kg per day,about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Withinthis range the dosage may be 0.005 to 0.05 mg/kg per day, 0.05 to 0.5mg/kg per day, 0.5 to 5.0 mg/kg per day, or 5.0 to 50 mg/kg per day.

For oral administration, the CCR9 inhibitor is preferably provided inthe form of tablets containing 1.0 to 1000 milligrams of the activeingredient, particularly 1.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, 25.0mg, 50.0 mg, 75.0 mg, 100.0 mg, 150.0 mg, 200.0 mg, 250.0 mg, 300.0 mg,400.0 mg, 500.0 mg, 600.0 mg, 750.0 mg, 800.0 mg, 900.0 mg, and 1000.0mg of the active ingredient for the symptomatic adjustment of the dosageto the patient to be treated.

The CCR9 inhibitor may be administered on a regimen of 1 to 4 times perday, preferably once or twice per day.

In the treatment of IBD such as Crohn's disease and UC, an appropriatedosage level of an anti-α4β7 integrin antibody provides an effectiveamount of the antibody or a formulation thereof to induce remission ofIBD in a human patient. In some embodiments, the therapeuticallyeffective amount of anti-α4β7 integrin antibody is sufficient to achieveabout 5 μg/ml to about 60 μg/ml mean trough serum concentration ofanti-α4β7 integrin antibody at the end of the induction phase, e.g.,about 5 μg/ml to about 60 μg/ml, about 10 μg/ml to about 50 μg/ml, about15 μg/ml to about 45 μg/ml, about 20 μg/ml to about 30 μg/ml, about 25μg/ml to about 35 μg/ml, or about 30 μg/ml to about 60 μg/ml mean troughserum concentration of anti-α4β7 integrin antibody at the end of theinduction phase.

Suitable dosages of antibody can be administered from about 0.1 mg/kg,about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, or about 10 mg/kg.

In some embodiments, the total dose amount is about 6 mg, about 10 mg,about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525mg, about 550 mg, about 575 mg, about 650 mg, or more.

In some embodiment, the induction phase is for at least about 2 weeks,at least about 3 weeks, at least about 4 weeks, at least about 5 weeks,at least about 6 weeks, at least about 7 weeks, at least about 8 weeks,at least about 9 weeks, or at least about 10 weeks of treatment.

The treatment regime during the induction phase can includeadministration of a high dose, frequent administrations, or acombination of a high dose and frequent administrations of the anti-α4β7integrin antibody or a formulation thereof. In some cases during theinduction phase, a dose is administered once per day, every other day,every two days, every three days, once per week, every 10 days, onceevery two weeks, once every three weeks or once a month.

In some embodiments, the induction dosing is provided once at initiationof treatment (day 0) and once at about two weeks after initiation oftreatment. The induction phase duration can be six weeks. In otherembodiments, the induction phase duration is six weeks and a pluralityof induction doses are administered during the first two weeks. Ininstances, when the human patient has severe IBD or is not responding toanti-TNFα therapy, the induction phase has longer duration than apatient who has mild to moderate IBD.

Also, in the treatment of IBD, an appropriate dosage level of ananti-α47β integrin antibody provides an effective amount of the antibodyor a formulation thereof to maintain remission of IBD in a humanpatient. As such, during the maintenance phase of the treatment, thetherapeutically effective amount of anti-α4β7 integrin antibody issufficient to achieve about 1 μg/ml to about 25 μg/ml mean steady statetrough serum concentration of anti-α4β7 integrin antibody during themaintenance phase, e.g., about 1 μg/ml to about 25 μg/ml, about 1 μg/mlto about 20 μg/ml, about 1 μg/ml to about 15 μg/ml, about 1 μg/ml toabout 10 μg/ml, about 1 μg/ml to about 5 μg/ml, about 5 μg/ml to about25 μg/ml, about 5 μg/ml to about 20 μg/ml, about 5 μg/ml to about 15μg/ml, about 5 μg/ml to about 10 μg/ml, about 15 μg/ml to about 25μg/ml, about 15 μg/ml to about 20 μg/ml, about 10 μg/ml to about 25μg/ml, about 10 μg/ml to about 20 μg/ml, about 10 μg/ml to about 15μg/ml, or about 20 μg/ml to about 25 μg/ml mean steady state troughserum concentration of anti-α4β7 integrin antibody at the end of theinduction phase.

The maintenance dose can be administered once a week, once every otherweek, once every three weeks, once every 4 weeks, once every 5 weeks,once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9weeks, or once every 10 weeks. In some embodiments during themaintenance phase, the same dosing amount is administered. In otherembodiments during the maintenance phase, one or more different dosingamounts are administered over the maintenance phase. Additionally,depending on the disease course, the dosing frequency can be increased.

The anti-α4β7 integrin antibody or formulation thereof can beadministered by injection, e.g., intravenous injection, intramuscularinjection, subcutaneous injection, intraarterial injection,intraperitoneal injection, intravitreal injection, and the like. If theformulation is in a solid or lyophilized form, the process ofadministering the antibody can include reconstituting the dryformulation into a liquid formulation. In some embodiments, the antibodyor formulation thereof can be administered topically, e.g., in a patch,cream, aerosol or suppository. In other embodiments, the topical routesof administration include nasal, inhalational or transdermaladministration.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, hereditary characteristics, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

The weight ratio of the CCR9 inhibitor described herein to the anti-α4β7integrin antibody of the present disclosure may be varied and willdepend upon the effective dose of each ingredient. Generally, aneffective dose of each will be used. Thus, for example, wherein a CCR9inhibitor is combined with an anti-α4β7 integrin antibody, the weightratio of the CCR9 inhibitor to the anti-α4β7 integrin antibody willgenerally range from about 1000:1 to about 1:1000, preferably about200:1 to about 1:200.

Combination therapy includes co-administration of the CCR9 inhibitor andthe anti-α4β7 integrin antibody, sequential administration of the CCR9inhibitor and the anti-α4β7 integrin antibody, administration of acomposition containing the CCR9 inhibitor and the anti-α4β7 integrinantibody, or simultaneous administration of separate compositions suchthat one composition contains the CCR9 inhibitor and another compositioncontains the anti-α4β7 integrin antibody.

Co-administration includes administering the CCR9 inhibitor of thepresent invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hoursof the anti-α4β7 integrin antibody of the present invention.Co-administration also includes administering simultaneously,approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30minutes of each other), or sequentially in any order. Moreover, the CCR9inhibitor and anti-α4β7 integrin antibody can each be administered oncea day, or two, three, or more times per day so as to provide thepreferred dosage level per day.

The combination therapy can be administered at an induction phase ormaintenance phase of the treatment regimen. In the induction phase, thecombination therapy can be administered at an effective amount to induceimmune tolerance to the antibody of the therapy, induce a clinicalresponse, and/or ameliorate one or more symptoms of IBD. Also, if duringthe maintenance phase, there is a return of one or more symptoms of IBDor if there is a relapse from remission of the disease, a patient can beadministered an amount corresponding to an induction phase treatment.During the maintenance phase, the combination therapy can beadministered at an effective amount to continue the response achieveduring the induction therapy and/or prevent the return of symptoms orrelapse of IBD.

In some embodiments, one or more additional active ingredients such asan anti-inflammatory compound, e.g., sulfasalazine, azathioprine,6-mercaptopurine, 5-aminosalicylic acid containing anti-inflammatories,a non-steroidal anti-inflammatory compound, and a steroidalanti-inflammatory compound; antibiotics commonly administered forcontrol of IBD, e.g., ciprofloxacin and metronidazole; or anotherbiologic agent, e.g., a TNFα antagonist can be administered inconjunction with the combination therapy disclosed herein.

D. Kits

In some aspects, provided herein are kits containing a CCR9 inhibitorand an anti-α4β7 integrin antibody disclosed herein that are useful fortreating a disease or disorder characterized by inflammation of thegastrointestinal tract such as IBD, including CD, UC and indeterminatecolitis. A kit can contain a pharmaceutical composition containing aCCR9 inhibitor compounds, e.g., a small molecule inhibitor of CCR9 and apharmaceutical composition containing an anti-α4β7 integrin antibody. Insome embodiments, the CCR9 inhibitor compound is vercirnon(Traficet-EN™) or CCX507. In some embodiments, the anti-α4β7 integrinantibody is vedolizumab. In some instances, the kit includes writtenmaterials e.g., instructions for use of the compound, antibody orpharmaceutical compositions thereof. Without limitation, the kit mayinclude buffers, diluents, filters, needles, syringes, and packageinserts with instructions for performing any methods disclosed herein.

IV. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1: Using Combination Therapy of a CCR9 Inhibitor and anAnti-α4β7 Integrin Antibody to Treat Inflammatory Bowel Disease

A. Introduction

Homing of circulating cells into various tissues is a highly coordinatedprocess involving specific chemokine receptors and cellular adhesionmolecules. Trafficking of cells into the gut requires chemokine receptorCCR9-mediated chemotaxis towards the chemokine known as CCL25. CCR9activation by CCL25 also triggers high affinity binding of cell surfaceα4β7 integrin with MAdCAM-expressing intestinal microvascularendothelium resulting in firm arrest and diapedesis into gut tissues.

Analysis of human colonic biopsies obtained from patients with colonicCrohn's disease showed strong positive correlation between CCR9 geneexpression and that of the genes for TNF-α, as well as α4 and β7integrins. These results showed that the regulation of genes implicatedin gut inflammation are highly associated and tightly regulated in thecolon. Vedolizumab, a humanized antibody against α4β7 integrin, wasrecently approved to treat patients with moderate to severe ulcerativecolitis and Crohn's disease. However, its effects in combination with anantagonist of the gut homing chemokine receptor CCR9 have not beenexplored.

B. Methods

Adhesion assays were performed as follows. Human lymphocytes wereisolated from total PBMCs and activated with α-CD3ε/α-CD28 (1 μg/mL; R&DSystems) in the presence of 1 μM retinoic acid (Sigma) and 1 ng/mL humanIL12 (R&D Systems). In vitro activated T cells were further expanded for5 days in the presence of 1 μM retinoic acid (RA) and 1 ng/mL humanIL12. The expanded cells were stained with an anti CCR9 APC conjugatedantibody (Cat. No. 248621; R&D Systems) and a conjugated anti-α4β7integrin antibody (Act-1). The stained cells were analyzed by flowcytometry. Expression of CCR9 and α4β7 integrin in the expanded T cellswas depicted as a 2-parameter scatterplot in FIG. 1A.

In vitro activated T cells were added to 96-well plate coated overnightwith 0.3 μg/mL MAdCAM-1-Fc fusion protein (R&D Systems) in the presenceof the following conditions: DMSO only, 500 ng human CCL25 (hCCL25; aCCR9 ligand) only, 1 μM CCX507 (a CCR9 small molecule inhibitor) only,or 500 ng hCCL25 and 1 μM CCX507. Adherent cells were quantified usingCyQUANT® Cell Proliferation Assays (Thermo Fisher). The results areprovided in FIG. 1B. The data shows that CCX507 limits the binding ofRA-differentiated human T cells to MAdCAM-1 which can bind α4β7 integrinand helps to direct lymphocyte trafficking.

In vivo pharmacodynamic assays were performed as follows. FIG. 2Adepicts the adoptive T cell transfer model used in the experiment. Adetailed description of the method is found in, e.g., Tubo et al., PLOSOne, 2012, 7(11):e50498. Briefly, CD8⁺ T cells isolated from donor OT-1TCR transgenic mice (B6.CD45.2). Leukocytes were isolated from thespleen and lymph node. The isolated CD8⁺ T cells were adoptivelytransferred into wild-type mice (B6. CD45.1). 1×10⁶-1×10⁷ naïve CD8⁺ Tcells were injected intraperitoneally into the recipient mouse.

24 hours post-transfer, the recipient CD45.1 mice were orally challengedwith cholera toxin alone or cholera toxin and ovalbumin. The choleratoxin challenged mice were used as a mouse model of intestinalinflammation. After 96 hours, leukocytes were isolated from the spleen,lymph nodes, small bowel, colon, blood and liver. The number ofdonor-derived CD8⁺ T cells in the small intestine was determined by flowcytometry 96 hours post-challenge. A detailed description of the methodis found in, e.g., Tubo et al., PLOS One, 2012, 7(11):e50498.

In addition, the cholera toxin challenged mice were administered witheither 5-30 mg/kg of CCX507 or a dose of an anti-α4β7 integrin antibody.The number of donor-derived (OT-1 derived) CD8⁺ intra-epitheliallymphocytes (IELs) were counted. The results are provided in FIG. 2B.

QuantiGene® Plex assays (Affymetrix) were performed as follows. Matchedileum and colon biopsies from Crohn's disease patients were snap frozenin liquid nitrogen. Samples were homogenized according to manufacturer'sprotocols. Expression of genes was analyzed using a customized 37-plexpanel (Affymetrix) and its expression was normalized to the housekeepinggene cyclophilin. FIG. 3 provides a comparison of expression levels ofselected genes compared to the level of CCR9 expression.

A mouse model of piroxicam accelerated colitis (chemically-inducedcolitis) was generated as follows. 7-week old Mdr1a^(−/−) mice and anFVB control strain were provided with 200 ppm piroxicam (Sigma) mixedwith powdered chow (food) in powdered diet feeding jars (Dyets, Inc.)for 10-12 days. Normal food was placed in wire bar lids for the durationof the experiment. Anti-α4β7 integrin blocking antibody (DATK32) wasadministered IP, q2d (via intraperitoneal injection every other day) at100 μg per mouse. Anti-TNFα antibody (XT3.11) was administered IP, qd(via intraperitoneal injection every day) at 300 μg per mouse during thefirst 11 days when piroxicam was added to the powdered chow. Anti-TNFαantibody (XT3.11) was administered IP, q2d at 300 μg per mouse afterpiroxicam was removed. CCX507 (a CCR9 small molecule inhibitor) wasadministered SC, qd (via subcutaneous injection every day) at 30 mg/kgfor the duration of the study. Mice in the study were observed andhandled as approved by IACUC. FIG. 4A depicts the experimental design.

FIG. 4B shows representative images of the colon of mice receiving avehicle control (1% HPMC) or a rat IgG2A isotype control, CCX507 (a CCR9small inhibitor molecule) alone, an anti-α4β7 integrin blocking antibodyalone, or CCX507 in addition to an anti-α4β7 integrin blocking antibody.Quantitative colon vs. weight ratios for the different treatmentconditions is depicted in FIGS. 4C and 4D. The combination therapy ofCCX507 and anti-α4β7 integrin blocking antibodies is shown in FIG. 4C.The combination therapy of CCX507 and anti-TNFα blocking antibodies isshown in FIG. 4D. Statistical analysis was performed using GraphPadPrism® (GraphPad Software) with the p-values represented in thefollowing manner: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Colon tissue, formalin fixed at the time of necropsy, was evaluated forhistopathology by an independent, blinded pathologist. A totalhistological score was derived from the combined histological scores ofInflammation, glandular epithelial loss and erosion. These scores werebased on the percentage of tissue affected in each section with a totalof 6 sections cut per mouse.

FIG. 5A shows representative images of both the proximal and distalcolon from individual mice, representative of the mean histopathologicalscore for each group. The combination of CCX507 in addition to anti-α4β7antibody is shown in the right panel.

The sum histological score for all mice in the study is represented inFIG. 5B. Statistical analysis was performed by GraphPad Prism with thep-values represented as *p<0.05, ****p<0.0001.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A kit for treating or reducing the development ofinflammatory bowel disease in a mammal, said kit comprising: (i) atherapeutically effective amount of a CCR9 chemokine receptor inhibitoror a pharmaceutically acceptable salt thereof; (ii) a therapeuticallyeffective amount of an anti-α4β7 integrin blocking antibody; and (iii)instructions for effective administration.
 2. The kit of claim 1,wherein the inflammatory bowel disease is Crohn's disease (CD) orulcerative colitis (UC).
 3. The kit of claim 1, wherein the anti-α4β7integrin blocking antibody is vedolizumab.
 4. The kit of claim 1,wherein the CCR9 chemokine receptor inhibitor is a compound havingformula (I) or a salt thereof:

where R¹ is selected from the group consisting of substituted orunsubstituted C₂₋₈ alkyl, substituted or unsubstituted C₁₋₈ alkoxy,substituted or unsubstituted C₁₋₈ alkylamino, and substituted orunsubstituted C₃₋₁₀heterocyclyl, and; R² is H, F, Cl, or substituted orunsubstituted C₁₋₈ alkoxy; or R¹ and R² together with the carbon atomsto which they are attached form a non-aromatic carbocyclic ring or aheterocyclic ring; R³ is H, substituted or unsubstituted C₁₋₈ alkyl,substituted or unsubstituted C₁₋₈ alkoxy, or halo; R⁴ is H or F; R⁵ isH, F, Cl, or —CH₃; R⁶ is H, halo, —CN, —CO₂R^(a), —CONH₂, —NH₂,substituted or unsubstituted C₁₋₈ alkyl, substituted or unsubstitutedC₁₋₈ alkoxy, or substituted or unsubstituted C₁₋₈ aminoalkyl; R^(a) is Hor substituted or unsubstituted C₁₋₈ alkyl; where R⁵ and R⁶ may togetherform a carbocyclic ring; L is a bond, —CH₂—, or —CH(CH₃)—; each of A¹,A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected from the groupconsisting of N, N—O, and —CR⁸—; where at least one and not more thantwo of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are N or N—O; R⁸ is eachindependently selected from the group consisting of H, halo, —CN, —OH,oxo, substituted or unsubstituted C₁₋₈ alkyl, substituted orunsubstituted C₁₋₈ alkoxy, and —NR²⁰R²¹, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl; and R²⁰ and R²¹ are each independently H, orsubstituted or unsubstituted C₁₋₈ alkyl.
 5. The kit of claim 4, whereone of A¹ or A² is N or N—O, and the remaining of A¹, A², A³, A⁴, A⁵,A⁶, A⁷, and A⁸ are —CR⁸—, where each R⁸ is selected independently. 6.The kit of claim 1, wherein the CCR9 chemokine receptor inhibitor is acompound having formula (II) or a salt thereof:

where R¹ is selected from the group consisting of substituted orunsubstituted C₂₋₈ alkyl, substituted or unsubstituted C₁₋₈alkoxy,substituted or unsubstituted C₁₋₈ alkylamino, and substituted orunsubstituted C₃₋₁₀ heterocyclyl; R² is H, F, Cl, or substituted orunsubstituted C₁₋₈ alkoxy; or R¹ and R² together with the carbon atomsto which they are attached form a non-aromatic carbocyclic ring or aheterocyclic ring; R³ is H, substituted or unsubstituted C₁₋₈ alkyl,substituted or unsubstituted C₁₋₈ alkoxy, or halo; R⁴ is H or F; R⁵ isH, F, Cl, or —CH₃; R⁶ is H, halo, —CN, —CO₂R^(a), —CONH₂, —NH₂,substituted or unsubstituted C₁₋₈ aminoalkyl, substituted orunsubstituted C₁₋₈ alkyl, or substituted or unsubstituted C₁₋₈ alkoxy;R^(a) is H or substituted or unsubstituted C₁₋₈ alkyl; where R⁵ and R⁶may together form a carbocyclic ring; L is a bond, —CH₂—, or —CH(CH₃)—;and Z is selected from the group consisting of:

and N-oxides thereof; where the Z group may be unsubstituted orsubstituted with 1 to 3 independently selected R⁸ substituents; each R⁸is independently selected from the group consisting of H, halo, —CN,—OH, oxo, substituted or unsubstituted C₁₋₈alkyl, substituted orunsubstituted C₁₋₈ alkoxy, and —NR²⁰R²¹, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl; and R²⁰ and R²¹ are each independently H,substituted or unsubstituted C₁₋₈ alkyl.
 7. The kit of claim 6, whereinthe inhibitor is a compound having formula (IIIa) or (IIIb) or a saltthereof:

where R¹ is selected from the group consisting of substituted orunsubstituted C₂₋₈ alkyl, substituted or unsubstituted C₁₋₈ alkoxy,substituted or unsubstituted C₁₋₈ alkylamino, and substituted orunsubstituted C₃₋₁₀ heterocyclyl; R² is H, F, Cl, or substituted orunsubstituted C₁₋₈ alkoxy; or R¹ and R² together with the carbon atomsto which they are attached form a non-aromatic carbocyclic ring or aheterocyclic ring; R³ is H, substituted or unsubstituted C₁₋₈ alkyl,substituted or unsubstituted C₁₋₈ alkoxy, or halo; R⁴ is H or F; R⁵ isH, F, Cl, or —CH₃; R⁶ is H, halo, —CN, —CO₂R^(a), —CONH₂, —NH₂,substituted or unsubstituted C₁₋₈ aminoalkyl, substituted orunsubstituted C₁₋₈ alkyl, or substituted or unsubstituted C₁₋₈ alkoxy;R^(a) is H or substituted or unsubstituted C₁₋₈ alkyl; or where R⁵ andR⁶ together with the carbon atoms to which they are attached form acarbocyclic ring; each R⁸ is independently selected from the groupconsisting of H, halo, —CN, —OH, oxo, substituted or unsubstituted C₁₋₈alkyl, substituted or unsubstituted C₁₋₈ alkoxy, and —NR²⁰R²¹,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted heterocyclyl; R²⁰ and R²¹are each independently H, or substituted or unsubstituted C₁₋₈ alkyl;and n is 0, 1, 2 or
 3. 8. The kit of claim 7, wherein the compound isselected from the group consisting of


9. The kit of claim 1, wherein the CCR9 chemokine receptor inhibitor is


10. The kit of claim 1, wherein the CCR9 chemokine receptor inhibitorand the anti-α4β7 integrin blocking antibody are formulated forsequential administration or concomitant administration.
 11. The kit ofclaim 10, wherein the CCR9 chemokine receptor inhibitor and theanti-α4β7 integrin blocking antibody are formulated for concomitantadministration.
 12. A kit for treating or reducing the development ofinflammatory bowel disease in a mammal, said kit comprising: (i) atherapeutically effective amount of vercirnon or a pharmaceuticallyacceptable salt thereof; (ii) a therapeutically effective amount of ananti-α4β7 integrin blocking antibody; and (iii) instructions foreffective administration.
 13. The kit of claim 12, wherein the CCR9chemokine receptor inhibitor and the anti-α4β7 integrin blockingantibody are formulated for sequential administration or concomitantadministration.
 14. The kit of claim 13, wherein the CCR9 chemokinereceptor inhibitor and the anti-α4β7 integrin blocking antibody areformulated for concomitant administration.
 15. The kit of claim 12,wherein the inflammatory bowel disease is Crohn's disease (CD) orulcerative colitis (UC).
 16. The kit of claim 12, wherein the anti-α4β7integrin blocking antibody is vedolizumab.
 17. A kit for treating orreducing the development of inflammatory bowel disease in a mammal, saidkit comprising: (i) a therapeutically effective amount of a CCR9chemokine receptor inhibitor or a pharmaceutically acceptable saltthereof; (ii) a therapeutically effective amount of an anti-TNFαblocking antibody; and (iii) instructions for effective administration.18. The kit of claim 17, wherein the inflammatory bowel disease isCrohn's disease (CD) or ulcerative colitis (UC).
 19. The kit of claim17, wherein the anti-α4β7 integrin blocking antibody is vedolizumab. 20.The kit of claim 17, wherein the CCR9 chemokine receptor inhibitor is